The complete chloroplast genome of an endangered plant Artemisia borotalensis (Asteraceae) and phylogenetic analysis

Abstract Artemisia borotalensis Poljakov is an endemic and endangered herb in China. In this study, we sequenced and analyzed the complete chloroplast genome of this species. Sequencing revealed the genome to be 151,179 bp in length, containing a large single copy region (82,862 bp), a small single copy region (18,377 bp), and a pair of inverted repeat regions (24,970 bp each). Our analyses demonstrated that it contained 133 genes, including 87 protein-coding genes, 37 transfer RNA genes, eight ribosomal RNA genes, and one pseudogene (ycf1). Furthermore, we found the genome to have an overall GC content of 37.4%. A phylogenetic analysis indicated that A. borotalensis and A. maritima clustered together as sister group to A. annua and A. fukudo clade.


Introduction
Artemisia borotalensis Poljakov 1954, an endemic plant in China, is a perennial herb from the family Asteraceae (Ling et al. 2011). In China, A. borotalensis is distributed over desert or semi-desert grassland in the Gobi and on gravel hillside areas in Xinjiang (Figure 1). Most Artemisia species have important medicinal, ecological, and economic value (Duffy and Mutabingwa 2006;Vall es et al. 2011). Song et al. (2021) reported that the essential oils contained in A. borotalensis can effectively kill cotton aphids and psyllids. However, A. borotalensis has become an endangered species due to its relatively narrow area of distribution and the disturbance of its original habitat destroyed by human activities. Therefore, using the genome skimming approach, we constructed the complete chloroplast genome of A. borotalensis, which will contribute to the conservation of this taxon's genetic resources and the understanding of its phylogenetic relationships.

Materials
An A. borotalensis specimen was collected from Xinjiang, China (46 7 0 25.6 00 N, 84 31 0 58 00 E; Altitude: 840 m). A. borotalensis is not a legally protected species in China, despite the decline in the size of its population. Collection of the sample followed the wild plant protection regulations of China. The voucher specimen was deposited in the Herbarium of the Institute of Botany, Chinese Academy of Sciences (PE, Xian-Chun Zhang, zhangxc@ibcas.ac.cn), with collection number ZC-451.

Methods
Total genomic DNA was extracted from approximately 100 mg of silica-dried leaf material using a modified CTAB method (Doyle and Doyle 1987). DNA extracts were fragmented to construct a 300 bp short-insert library and sequenced with 2 Â 150 bp paired-end reads on DNBSEQ TM technology platforms at Beijing Genomics Institute (Shenzhen, China). The raw reads were filtered using Trimmomatic 0.35 (Bolger et al. 2014) to remove adapters and low-quality bases. Then, about 2.5 Gb of clean reads were assembled using GetOrganelle v. 1.7.1 (Jin et al. 2020). The chloroplast genomes were annotated with GeSeq (Tillich et al. 2017), before being manually adjusted with Geneious v. 9.1.7 (Kearse et al. 2012). Finally, the annotated chloroplast genomes were submitted to GenBank using Bankit (Accession number: ON964524), and Organellar Genome Draw (OGDRAW) (Lohse et al. 2007) was used to illustrate the circular genome map.
To investigate the phylogenetic position of A. borotalensis, we reconstructed its phylogenetic relationships based on 16 complete chloroplast genomes from 10 Artemisia species and used the closely related species Ajania pacifica (NC_050690) as an outgroup. The 17 complete chloroplast genomes (after removing one inverted repeat region) were aligned using the default parameters in MAFFT v. 7 (Katoh and Standley 2013). According to the Bayesian information criterion (BIC), the most appropriate substitution models, estimated using jModelTest2 (Darriba et al. 2012), were TVM þ I þ G for the complete chloroplast genome sequences. Maximum likelihood (ML) analyses were then conducted using RAxML-HPC v. 8 (Stamatakis 2014) with 1000 bootstrap replicates. The evaluation of the most appropriate substitution models and the construction of the phylogenetic tree were both carried out on the CIPRES Science Gateway portal (Miller et al. 2010).

Results
The sequenced chloroplast genome of A. borotalensis was found to possess a typical quadripartite structure, which consisted of large single copy (LSC), small single copy (SSC), and inverted repeat (IRa and IRb) regions (Figure 2). The complete chloroplast genome was 151,179 bp in length, of which the LSC, SSC, and IR were 82,862 bp, 18,377 bp and 24,970 bp in length, respectively. In total, 133 genes were identified, including 87 protein-coding genes, 37 transfer RNA genes, eight ribosomal RNA genes, and one pseudogene (ycf1). The overall GC content of the chloroplast genome was shown to  be 37.4%. Comparison of the A. borotalensis chloroplast genome to previously published data revealed a high level of gene synteny with one publicly available genome data sets from A. maritima (Shahzadi et al. 2020). Furthermore, phylogenetic analysis showed that A. borotalensis and A. maritima clustered together as sister group (with 100% bootstrap support) to A. annua and A. fukudo clade (Figure 3).

Discussion and conclusions
The chloroplast genome of A. borotalensis sequenced in this study was consistent with those of other previously published Artemisia taxa in terms of gene structure, number, and GC content , suggesting that the chloroplast genome of this taxon is relatively evolutionarily conserved. Moreover, the phylogenetic results confirmed the classification of A. borotalensis within the Artemisia subgenus Seriphidium based on its morphological traits, which has not been addressed in previous phylogenetic studies due to sampling limitations (Malik et al. 2017). In future, our findings will contribute to the development and conservation of the genetic resources of Artemisia and lay the foundation on which to develop a further understanding the phylogenetic relationships between members of this genus.

Author contributions
Ying Feng was mainly responsible for the study design. Guangzhao Jin conducted the analysis of data and drafted the manuscript. Zhibin Wen and Feng Song helped perform the analysis with constructive discussions and revised it critically for intellectual content. All authors approve the version to be published and agree to be accountable for all aspects of the work.

Regulation statement
All materials used in the study were collected in public areas of China and strictly follows the wild plant protection regulations of China. Voucher specimens were prepared and deposited at the Herbarium of the Institute of Botany, Chinese Academy of Sciences (PE).

Disclosure statement
No potential conflict of interest was reported by the author(s).